WO2013172358A1 - Composé fonctionnel, assemblage moléculaire contenant un composé fonctionnel, composition contenant un assemblage moléculaire, trousse et utilisation de l'assemblage moléculaire, de la composition ou du trousse - Google Patents

Composé fonctionnel, assemblage moléculaire contenant un composé fonctionnel, composition contenant un assemblage moléculaire, trousse et utilisation de l'assemblage moléculaire, de la composition ou du trousse Download PDF

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WO2013172358A1
WO2013172358A1 PCT/JP2013/063459 JP2013063459W WO2013172358A1 WO 2013172358 A1 WO2013172358 A1 WO 2013172358A1 JP 2013063459 W JP2013063459 W JP 2013063459W WO 2013172358 A1 WO2013172358 A1 WO 2013172358A1
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lipid
group
molecular assembly
xhr
dendron
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PCT/JP2013/063459
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English (en)
Japanese (ja)
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河野　健司
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公立大学法人大阪府立大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a functional compound, a molecular assembly containing the compound, a composition and kit containing them, and uses thereof.
  • DDS drug delivery system
  • DDS for the purpose of improving target directivity of drugs, construction of so-called temperature-responsive carriers having a function of releasing drugs in response to temperature has been attempted. Specifically, an attempt has been made to use, as a carrier, a molecular assembly that shows a vesicle shape when not heated, but changes its shape when heated. After the drug is held inside the vesicle and administered into the living body, the structure of the molecular assembly is greatly changed by selectively heating a desired site such as an affected part, for example.
  • Non-patent Document 1 Attempts have been made to develop technology for releasing drugs only in More specifically, an attempt to use a molecular assembly composed of a compound in which a lipid having a polyamide dendron structure (polyamide dendron lipid) is modified with a temperature-responsive isobutylamide (IBAM) group for the above purpose. has been reported (Non-patent Document 1).
  • HLA major histocompatibility complex
  • HLA which is a human MHC
  • a peptide capable of acting on HLA Human Leukocyte Antigen
  • an immune-related disease can be treated by controlling HLA function.
  • cancer can be selectively treated by promoting antigen presentation by HLA and activating immunity.
  • the antigen is encapsulated in a liposome that has been modified with a pH-responsive polymer and can acquire a fusion ability with a lipid membrane in accordance with a change in pH, and then introduced into a dendritic cell, whereby the antigen is It has been reported that cell-mediated immunity was induced by delivery of the protein to the cytosol (Non-patent Document 2). More specifically, the liposome is once taken up into a cell by endocytosis, fuses with the endosome in response to the pH in the endosome, and finally releases the inclusion (antigen) into the cytosol.
  • Kenji Kono et al. “Thermosensitive Molecular Assembly from Poly (amidoamine) Dendron-Based Lipids”, “Angewandte Chemie International, 50 Years” 6332-6336 Eiji Yuba et al., “PH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic protein for activation of Biomass, 31”. 943-951
  • the first object of the present invention is to improve the balance of (1) temperature responsiveness, (2) in vivo stability and (3) biocompatibility in a temperature responsive carrier that can be used for the above purpose. .
  • the present invention selectively delivers a target substance, specifically, a physiologically active substance, to an intracellular organelle of any one of (1) early endosome, (2) late endosome, and (3) lysosome.
  • the second problem is to provide a means that can be released by the above-mentioned method, that is, to provide a carrier as the means, a method of using the carrier, and the like.
  • a molecular assembly can be formed using a compound obtained by modifying a polyamide dendron lipid with a hydrocarbon group containing an oligoethylene glycol structure, which is expected to be excellent in biocompatibility.
  • the present inventors have found that there is difficulty in in vivo stability. Therefore, the present inventors formed a molecular assembly using a lipid containing a polyethylene glycol structure in addition to the above compound. In such a molecular assembly, (1) temperature responsiveness, (2) in vivo It has been found that the balance between stability and (3) biocompatibility is improved compared to the conventional one.
  • the present inventors have intensively studied to solve the second problem.
  • the above problem can be solved by using a compound obtained by modifying a predetermined dendron lipid with a pH-responsive polymer. More specifically, it is as follows.
  • a desired substance is encapsulated in a liposome composed of the above compound and then introduced into a cell, (1) it is efficiently incorporated into the initial endosome by endocytosis.
  • the liposome has a property of releasing inclusions in response to a specific pH range (response pH range), and it is easy to adjust the response pH range.
  • the present invention has been completed by the inventors of the present invention based on the above findings, and has been completed and includes the following embodiments.
  • An embodiment of the invention (first invention) as a solution to the first problem is as follows.
  • Item 1. (A) a compound represented by any of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure.
  • R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom, and X represents —CH 2 CH 2 CONHCH 2 CH 2 N—. Show. ).
  • Item 2. The molecular assembly according to Item 1, wherein R 3 and R 4 are represented by the following formula (I):
  • Item 3. The molecular assembly according to Item 1 or 2, wherein the lipid (B) is represented by the following formula (II):
  • Y is a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 5 to 200, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • Item 4. The molecular assembly according to any one of Items 1 to 3, which is used for introducing a physiologically active substance into a cell.
  • Item 5. The molecular assembly according to any one of Items 1 to 3, which is used for treating a disease by introducing a physiologically active substance into a cell.
  • Item 6. Item 4. A composition containing the molecular assembly according to any one of Items 1 to 3.
  • Item 7. Item 7.
  • Item 8. Use of the molecular assembly, composition or kit according to any one of Items 1 to 7 in a method for introducing a physiologically active substance into a cell.
  • Item 9. Item 8.
  • An embodiment of the invention as means for solving the second problem is as follows.
  • Item 1 Compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2
  • R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10 carbon atoms (which may contain a cyclic structure, and one or more carbon atoms may be substituted with a hetero atom).
  • the pH-responsive carboxyl group-containing hydrocarbon group is (A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure that may have a branch, Item 3. The compound according to Item 1 or 2.
  • Item 5. The composition according to Item 4, comprising a phospholipid.
  • Item 6. The composition according to Item 4 or 5, which is used for introducing a physiologically active substance into cells.
  • Item 7. Item 6.
  • Item 8. Item 6.
  • Item 9. Item 6.
  • Item 10. Item 10.
  • Item 11 Use of the compound, composition or kit according to any one of Items 1 to 9 in a method for introducing a physiologically active substance into a cell.
  • Item 11. A method for introducing a physiologically active substance into a cell, comprising a step of introducing the compound, composition or kit according to any one of Items 1 to 9 into the cell together with the physiologically active substance.
  • a temperature-responsive carrier used for the purpose of improving the target directivity of a physiologically active substance that can be administered to a living body as a drug, comprising (1) temperature responsiveness, (2) A carrier having an improved balance between in vivo stability and (3) biocompatibility can be provided.
  • the carrier by using the carrier, it is possible to provide a method for delivering a physiologically active substance with improved target directivity and reduced harm that can be given to a living body.
  • the target substance can be selectively delivered to any desired intracellular organelle of (1) early endosome, (2) late endosome, and (3) lysosome.
  • a physiologically active substance that can become an antigen or generate an antigen when used as a physiologically active substance, cell immunity can be achieved through delivery of the substance to intracellular organelles by targeting the antigen-presenting cell. It has an excellent effect that can be induced.
  • Differential scanning calorimetry of MDEG-DL-G1 assembly dispersion, MDEG-DL-G2 assembly dispersion, and MDEG-DL-G1-U2 assembly dispersion (10 mM phosphoric acid 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart.
  • Differential scanning calorimetry of EDEG-DL-G1 assembly dispersion, EDEG-DL-G2 assembly dispersion, and EDEG-DL-G1-U2 assembly dispersion (10 mM phosphate 140 m NaCl aqueous solution, pH7.4) It is a measurement (DSC) chart.
  • EDEG-DL-G1-2C 18 / PEG-Chol (95/5) and MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregates both have a cloud point at about 40 ° C.
  • FIG. 5 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UCG1.25, SCG1.25 and EYPC are respectively MGlu- DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 / EYPC (25/75, mol / mol) and liposomes with EYPC as components).
  • UG1.25, UCG1.25, SCG1.25 and EYPC are respectively MGlu- DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 -U2 / EYPC (25/75, mol / mol), CHex-DL-G1-2C 18 / EYPC
  • FIG. 6 is a graph showing the results of plotting pyranine release (%) against pH after incubation of various liposomes at 37 ° C. for 15 minutes (UMG1.25, UMG1.40 and EYPC are respectively MGMG-DL-U2 / Liposomes with EYPC mole ratios of 25, 40 and 0 are shown). It is a graph showing the results of plotting the release of pyranine (%) against pH after incubating liposomes containing CHexDL-U2 / EYPC for 15 minutes at 37 ° C (UCG1.25, UCG1.10 and EYPC are , Liposomes with CHexDL-U2 / EYPC molar ratios of 25, 10 and 0, respectively).
  • CHex-DL-2C 18 -U2 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 -U2 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • CHex-DL-2C 18 / EYPC 25/75, mol / mol
  • EYPC The pyranine release behavior at various pH 7.4 encapsulating pyranine is shown (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • EYPC UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • EYPC UMCG1.1525, UMCG1.2020 and UMG1.40 are respectively EYPC liposome, MGlu-G1-2C 18 -U2 / CHex-G1- 2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposome, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20/60, mol / mol / mol) Liposomes and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposomes).
  • Various liposome encapsulated pyranine shows pyranine release behavior after 15 minutes incubation at various pH (EYPC, UMCG1.1525, UMCG1.2020 and UMG1.40 each EYPC liposomes, MGlu-G1-2C 18 - U2 / CHex-G1-2C 18 -U2 / EYPC (15/25/60, mol / mol / mol) Liposomes, MGlu-G1-2C 18 -U2 / CHex-G1-2C 18 -U2 / EYPC (20/20 / 60, mol / mol / mol) liposome and CHex-G1-2C 18 -U2 / EYPC (40/60, mol / mol / mol) liposome).
  • the molecular assembly of the present invention comprises: (A) a compound represented by any one of the following formulas DL-G1 to DL-G4; and (B) a molecular assembly DL-G1: R 1 R 2 NX (XHR containing a lipid containing a polyethylene glycol structure 3 ) XHR 4 DL-G2: R P R 2 NX (X (XHR 3) XHR 4) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, and R 3 and R 4 are hydrocarbons containing the same or different oligoethylene glycol structure. Containing groups, R 1 to R 4 may
  • X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms. However, one hydrogen atom may be substituted with a hydrophobic amino acid or an alkyl group.
  • hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine.
  • alkyl group include a methyl group, an ethyl group, a normal propyl group, an isopropyl group, and a cyclopropyl group.
  • R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
  • the long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
  • the number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain.
  • the number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
  • the long chain hydrocarbon group may have a branch.
  • the main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
  • the long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a phenyl group, a cholesteryl group, a cyclohexyl group etc. are mentioned. Particularly preferred is a cholesteryl group.
  • the long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom.
  • the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom.
  • the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
  • the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
  • oxygen-containing hydrocarbon group examples include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond.
  • oxygen-containing hydrocarbon group having a carbonyl bond examples include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
  • nitrogen-containing hydrocarbon group examples include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
  • sulfur-containing hydrocarbon group examples include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
  • the long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • the unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
  • the type of unsaturated bond may be a double bond or a triple bond.
  • a double bond is preferred.
  • the number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
  • the double bond may be a cis-type double bond or a trans-type double bond.
  • a cis type double bond is preferred.
  • unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
  • unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
  • a 9-octadecenyl group (oleyl group) is more preferable.
  • Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
  • Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example.
  • the structure may further include a structure as exemplified above.
  • the group having the structure as exemplified above as a preferred example may further have at least one substituent.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a phenylene group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • hydrocarbon group containing oligoethylene glycol structure is oligoethylene glycol. It only needs to contain a structure, and may further contain other structures. In the oligoethylene glycol-containing hydrocarbon group, other structures may be interposed between the oligoethylene glycol structures, or the oligoethylene glycol structure may be interposed between the other structures. Good.
  • the other structure may be a linear structure which may have a branch.
  • a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
  • the other structure may be a ring structure.
  • the cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group.
  • a cyclohexyl group is particularly preferable.
  • one or more carbon atoms may be substituted with a heteroatom.
  • the oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • the oligoethylene glycol structure has 1 to 10 oxyethylene units continuously, in other words, without any other structure interposed. Structure is mentioned.
  • the oligoethylene glycol structure preferably has 1 to 4 oxyethylene units in succession.
  • the oligoethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • a structure having two or more divided oxyethylene units in other words, through another structure, a total of 1 Examples include structures having up to 10 oxyethylene units. In this case, those having a total of 1 to 4 oxyethylene units are preferred.
  • the branched chain of the oligoethylene glycol-containing hydrocarbon group may further have an oligoethylene glycol structure.
  • Oligoethylene glycol-containing hydrocarbon group is preferably represented by the following formula (I):
  • n represents an integer of 1 to 10 and R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)).
  • R 5 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom)
  • Specific examples of the oligoethylene glycol-containing hydrocarbon group include those represented by the following formula (III).
  • n an integer of 1 to 10
  • lipid (B) Containing Polyethylene Glycol Structure only needs to contain a polyethylene glycol structure, and as long as the effects of the present invention are exhibited, It is not limited.
  • the lipid (B) containing a polyethylene glycol structure is divided into a part containing a polyethylene glycol structure and a lipid part.
  • the part containing the polyethylene glycol structure only needs to contain a polyethylene glycol structure, and may further contain other hydrocarbon structures.
  • another hydrocarbon structure may be interposed between the polyethylene glycol structures, or the polyethylene glycol structure may be interposed between the other hydrocarbon structures.
  • the other hydrocarbon structure may have a branch or a straight chain structure.
  • Such a structure is not particularly limited, and examples thereof include those having a straight chain portion having 1 to 10 carbon atoms which may have a branch having 1 to 10 carbon atoms.
  • the other hydrocarbon structure may be a cyclic structure.
  • the cyclic structure is not particularly limited, and examples thereof include a cyclopropyl group, an epoxy group, a cycloheptyl group, a cyclobutyl group, a phenyl group, a cholesteryl group, and a cyclohexyl group.
  • a cyclohexyl group is particularly preferable.
  • one or more carbon atoms may be substituted with a hetero atom.
  • the polyethylene glycol structure is not particularly limited as long as the effects of the present invention can be achieved.
  • a structure having 3 to 200 oxyethylene units continuously in other words, without any other structure interposed therebetween.
  • the polyethylene glycol structure preferably has 3 to 200 oxyethylene units in succession, more preferably 10 to 100.
  • the lipid portion is not particularly limited as long as the effect of the present invention is exhibited.
  • Examples of the lipid moiety include a hydrocarbon group having 10 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • the hydrocarbon group may be saturated or unsaturated, and may have a cyclic structure.
  • lipid portion examples include phospholipids such as cholesterol and phosphatidylethanolamine, glycolipids, sphingolipids, long-chain fatty acids such as arachidonic acid, diacylglycerol, and the like.
  • the lipid (B) containing a polyethylene glycol structure is preferably represented by the following formula (II)
  • Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a heteroatom), and n represents any integer of 10 to 20, and R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • R 6 represents a hydrocarbon group having 1 to 10 carbon atoms (one or more carbon atoms may be substituted with a hetero atom).
  • Y is a hydrocarbon group having 5 to 50 carbon atoms (one or more carbon atoms may be substituted with a hetero atom), and n represents any integer of 10 to 20) .
  • the molecular assembly of the present invention is a molecular assembly containing a compound (A) and a lipid (B).
  • the molecular aggregate refers to an aggregate of at least the compound (A) and the lipid (B).
  • the molecular assembly of the present invention has a vesicle shape or an inverted hexagonal shape. Whether the molecular assembly is a vesicle shape or an inverted hexagonal shape can be confirmed using an atomic force microscope (AFM). If a spherical molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has a vesicle shape. Although not particularly limited, for example, a sphere having a particle size of about 150 to 200 nm can be observed as a vesicle-shaped molecular assembly. On the other hand, if a rod-like molecular assembly is observed by AFM observation, it can be evaluated that the molecular assembly has an inverted hexagonal shape.
  • the compound (A) of the present invention exhibits temperature responsiveness due to the action of the oligoethylene glycol structure.
  • the molecular assembly of the present invention has a vesicle shape under an environment of a predetermined temperature, but changes to an inverted hexagonal shape when the temperature of the environment increases.
  • the vesicle-shaped molecular assembly of the present invention forms a lipid bilayer in an aqueous solution so that the R 1 and R 2 sides face the inside and the R 3 and R 4 sides face the outside. Since the surface of this vesicle-shaped molecular assembly is hydrophilic, it is weakly adsorbed on the cell surface when administered in vivo. A desired hydrophobic substance can be held inside the lipid bilayer membrane of the vesicle-shaped molecular assembly.
  • the reverse hexagonal-shaped molecular assembly of the present invention has a hydrophobic surface and is easily taken up into cells by endocytosis.
  • a vesicle-shaped molecular assembly is administered to a living body in a state where a desired hydrophobic substance is held inside the lipid bilayer membrane, and the environmental temperature of the desired site among the sites to which the molecular assembly is delivered is set.
  • the substance can be selectively introduced into the cell at the site by raising and changing to an inverted hexagonal shape.
  • the molecular assembly of the present invention may be in a dry state or a frozen state.
  • each component containing the compound (A) and lipid (B) is once dissolved in an organic solvent such as chloroform and then dried under reduced pressure using an evaporator or a spray dryer. It can be manufactured by spray drying.
  • the compound (A) of the present invention can be obtained, for example, by adding R 3 and R 4 to a polyamide dendron (DL) obtained as follows.
  • DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively.
  • DL-G0 is the 0th generation and does not have a dendron structure.
  • R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
  • R ⁇ 1 > and R ⁇ 2 > is a saturated hydrocarbon group
  • Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material.
  • the polyamidoamine dendron denoted as DL-G1-2C 18.
  • R 3 and R 4 can be performed as follows, for example, as shown in the following formula.
  • MDEG methoxydiethylene glycol
  • EDEG ethoxydiethylene glycol
  • composition of the present invention further contains other components in addition to the molecular assembly of the present invention.
  • an aqueous solvent (dispersion medium) is contained.
  • other components include water, sugar aqueous solutions such as glucose, lactose, and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, phosphate buffer, and citrate buffer. Liquid, buffer solution such as phosphate buffered saline, physiological saline, medium for cell culture, and the like.
  • an aqueous solution of sugar when freeze-preserving or spray-drying preservation, an aqueous solution of sugar can be used, and when storing frozen, an aqueous solution of sugar or aqueous polyhydric alcohol can be used for effective preservation.
  • the concentration of these aqueous solvent additives is not particularly limited.
  • an aqueous sugar solution 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable.
  • a polyhydric alcohol aqueous solution 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable.
  • the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
  • the concentration of the molecular assembly of the present invention in the aqueous solvent is not particularly limited, but is preferably 0.01 mM to 100 mM, more preferably 0.1 mM to 10 mM.
  • the form in which the molecular assembly of the present invention is dispersed in an aqueous solvent is obtained by adding the dried lipid mixture to an aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like.
  • it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should
  • examples of a method for further drying the molecular assembly dispersed in the aqueous solvent include ordinary freeze drying and spray drying.
  • a sugar aqueous solution preferably a sucrose aqueous solution or a lactose aqueous solution may be used.
  • the molecular assembly can be stored for a long period of time, and when an aqueous solution containing a desired substance is added to the dried molecular assembly. This is preferable because the substance is efficiently retained in the molecular assembly.
  • the molecular assemblies and compositions of the present invention are preferably each used to introduce a desired hydrophobic substance into a cell.
  • the hydrophobic desired substance is not particularly limited, and examples thereof include physiologically active substances such as low molecular weight compounds, peptides, lipids, hormones, proteins, and nucleic acid derivatives.
  • physiologically active substances such as low molecular weight compounds, peptides, lipids, hormones, proteins, and nucleic acid derivatives.
  • various anticancer agents such as doxorubicin, hormone preparations, and antigen molecules for cancer immunotherapy such as WT1 peptide and its derivatives can be used.
  • the molecular assembly and composition of the present invention may be directly introduced into the individual to be treated together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases.
  • the molecular assembly or composition of the present invention may be administered to an individual.
  • the means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred.
  • the dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like.
  • Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
  • the blending ratio of the desired hydrophobic substance and the compound (A) of the present invention is such that the compound (A) of the present invention is 1 to 1000 parts by weight, preferably 10 to 500 parts by weight per 1 part by weight of the substance. Preferably 10 to 100 parts by weight are used.
  • Second invention Compound of the Present Invention
  • the compound of the present invention is a compound DL-G1: R 1 R 2 NX (XHR 3 ) XHR 4 represented by any of the following formulas DL-G1 to DL-G4 DL-G2: R 1 R 2 NX (X (XHR 3 ) XHR 4 ) 2 DL-G3: R 1 R 2 NX (X (X (XHR 3 ) XHR 4 ) 2 ) 2 DL-G4: R 1 R 2 NX (X (X (X (XHR 3 ) XHR 4 ) 2 ) 2 ) 2 (Wherein R 1 and R 2 are the same or different and represent a saturated or unsaturated long chain hydrocarbon group, R 3 and R 4 represent the same or different pH-responsive carboxyl group-containing hydrocarbon group, R 1 to R 4 may contain a cyclic structure, and one or more carbon atoms are heteroatoms And X represents —CH 2 CH 2 CONHCH 2 CH 2 N—.
  • X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms, but one hydrogen atom is a hydrophobic amino acid. May be substituted.
  • hydrophobic amino acids include leucine, valine, isoleucine, norleucine, phenylalanine and tyrosine.
  • R 1 and R 2 for R 1 and R 2 are long chain, saturated or unsaturated hydrocarbon group.
  • the long-chain hydrocarbon group may be naturally derived, may be modified from naturally derived, or may be artificially synthesized.
  • the number of carbon atoms of the long-chain hydrocarbon group is not limited as long as the main chain is a long chain.
  • the number of carbon atoms in the main chain is preferably 8 to 30, more preferably 10 to 22, and still more preferably 12 to 20.
  • the long chain hydrocarbon group may have a branch.
  • the main chain is determined based on the IUPAC nomenclature, or when it is difficult or impossible, it is determined based on a similar method.
  • the long chain hydrocarbon group may have a cyclic structure. Although it does not specifically limit as a cyclic structure, For example, a cyclohexyl group, a phenyl group, a pyridyl group, a naphthyl group, etc. are mentioned. A cyclohexyl group is particularly preferable.
  • the long chain hydrocarbon group may be one in which at least one carbon atom is substituted with a hetero atom.
  • the hetero atom means an oxygen atom, a nitrogen atom or a sulfur atom.
  • the long chain hydrocarbon group includes an oxygen-containing hydrocarbon group, a nitrogen-containing hydrocarbon group, and a sulfur-containing hydrocarbon group.
  • the hydrocarbon group means an alkyl group, an alkenyl group or an alkynyl group.
  • oxygen-containing hydrocarbon group examples include an oxygen-containing hydrocarbon group having at least one bond selected from the group consisting of an ether bond and a carbonyl bond.
  • oxygen-containing hydrocarbon group having a carbonyl bond examples include a group having at least one structure selected from the group consisting of aldehyde, ketone, carboxylic acid, ester, amide, enone, acid chloride, or anhydride. Can be mentioned.
  • nitrogen-containing hydrocarbon group examples include a nitrogen-containing hydrocarbon group having at least one structure selected from the group consisting of nitrile, amine, amide and imide.
  • sulfur-containing hydrocarbon group examples include thiol, thioether, thioacetal, sulfide, disulfide, dithiocarboxylic acid, thioester, thioketone, thioaldehyde, thiocarbamate, thiourethane, phosphine sulfide, thiophosphate, thiophosphonate, sulfonate, sulfone and And sulfur-containing hydrocarbon groups having at least one bond selected from the group consisting of sulfonamides.
  • the long chain hydrocarbon group may further have at least one substituent in the group having the structure as exemplified above.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • the unsaturated long chain hydrocarbon group is preferably a hydrocarbon group having an unsaturated bond in the main chain and the main chain having a chain structure.
  • the type of unsaturated bond may be a double bond or a triple bond.
  • a double bond is preferred.
  • the number of unsaturated bonds should just be at least 1 or more, and is not limited. Moreover, you may have both a double bond and a triple bond. It preferably has one double bond.
  • the double bond may be a cis-type double bond or a trans-type double bond.
  • a cis type double bond is preferred.
  • unsaturated long-chain hydrocarbon groups include hexadecenyl, octadecenyl, octadecadienyl, octadecatrienyl, icosatrienyl, icosatetraenyl, octadecatrienyl, icosapentaenyl, or docosa A hexaenyl group is preferred.
  • unsaturated long chain hydrocarbon groups include 9-hexadecenyl group, 9-octadecenyl group (oleyl group), 12-octadecadienyl group, 6,9,12-octadecatrienyl group, 8,11,14- Eicosatrienyl group, 5,8,11,14-icosatetraenyl group, 9,12,15-octadecatrienyl group, 5,8,11,14,17-icosapentaenyl group or 4,7,13 , 16,19-docosahexaenyl group is more preferred.
  • a 9-octadecenyl group (oleyl group) is more preferable.
  • Preferred examples of the unsaturated long-chain hydrocarbon group include those in which at least one carbon atom is further substituted with a hetero atom in the group having the structure exemplified above as a preferred example.
  • Preferred examples of the unsaturated long chain hydrocarbon group are specific to oxygen-containing hydrocarbon groups, nitrogen-containing hydrocarbon groups, or sulfur-containing hydrocarbon groups on the basis of the group having the structure exemplified above as a preferred example.
  • the structure may further include a structure as exemplified above.
  • the group having the structure as exemplified above as a preferred example may further have at least one substituent.
  • substituent those showing hydrophobicity are preferable.
  • substituent include a phenyl group, a cholesteryl group, and a pyrenyl group.
  • a cholesteryl group is particularly preferable.
  • pH-responsive carboxyl group-containing hydrocarbon group may contain a cyclic structure.
  • one or more carbon atoms may be substituted with a hetero atom.
  • the pH-responsive carboxyl group-containing hydrocarbon group is not particularly limited, but preferably has 2 to 20 carbon atoms, more preferably 2 to 10 carbon atoms, and still more preferably 2 to 6 carbon atoms.
  • the hetero atom is counted as a carbon atom.
  • the pH-responsive carboxyl group-containing hydrocarbon group is preferably represented by the following formula (I)
  • R 5 represents a saturated or unsaturated hydrocarbon group having 1 to 10, preferably 1 to 5, more preferably 1 to 3 carbon atoms (which may contain a cyclic structure and has one or more carbon atoms). Carbon atoms may be substituted with heteroatoms).
  • the pH-responsive carboxyl group-containing hydrocarbon group is more preferably (A) contains a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group, or (B) one or more carbon atoms having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a heteroatom). It is a chain structure which may have a branch.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A) those containing a cyclohexyl group are preferable.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (A) include those represented by the following formula (II).
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has one or more branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) is more preferably 1 to 2 having 1 or 2 carbon atoms (one or more carbon atoms may be substituted with a hetero atom). It is a chain structure which may have the following branches.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to the above (B) more preferably has 1 to 2 branches having 1 carbon atom (the carbon atom may be substituted with a hetero atom). It may be a chain structure.
  • the pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) has one branch having 1 carbon atom (the carbon atom may be substituted with a heteroatom). It is a good chain structure.
  • pH-responsive carboxyl group-containing hydrocarbon group corresponding to (B) above include those represented by the following formula (III).
  • the compound of the present invention forms a vesicle in an aqueous solution by forming a lipid bilayer with the R 1 and R 2 sides facing inward and the R 3 and R 4 sides facing outward. To do.
  • This vesicle is excellent in the ability to penetrate into cells.
  • a physiologically active substance or the like can be encapsulated in the vesicle. Therefore, the compound of the present invention can be used for introducing a physiologically active substance into cells.
  • the compound of the present invention exhibits pH responsiveness due to the action of a pH responsive carboxyl group-containing hydrocarbon group.
  • PH responsiveness means that a vesicle constituted by the compound of the present invention is destabilized under an environment of a predetermined pH and releases inclusions.
  • the compound of the present invention is derived from a pH-responsive carboxyl group-containing hydrocarbon group and has a carboxyl group (A) that can exhibit a negative charge when protons dissociate.
  • the compound of this invention has the tertiary amine (B) of the polyamide dendron which can receive a protonation. Therefore, in the compound of the present invention, when the pH is continuously changed from the basic side to the acidic side, the states of the carboxyl group (A) and the tertiary amine (B) on the left change as follows. It will follow.
  • the ⁇ potential of the compound is considered to change from a negative value to a positive value while continuously changing from the basic side to the acidic side.
  • the carboxyl group is protonated, the surface of the vesicle becomes hydrophobic, and further, the internal tertiary amine is protonated, resulting in electrostatic repulsion between molecules. Thereby, it is considered that the vesicle is destabilized and the inclusion is released.
  • the compound of the present invention exhibiting desired pH responsiveness can be provided.
  • foreign substances taken into cells via phagocytosis are first retained in (1) intracellular organelles called early endosomes, and eventually, early endosomes change to (2) late endosomes. . If kept in the late endosome, the foreign substance can finally be delivered to an intracellular organelle called (3) lysosome. And (1) the early endosome, (2) the late endosome, and (3) the interior of the lysosome have different pH values of about pH 6.2, pH 6.0 to 5.0, and pH 5.0 to 4.0, respectively. ing.
  • the compound of the present invention when it is used as a vesicle, it is stabilized in a pH range higher than, for example, pH 6.2, and is designed to be destabilized only at about pH 6.2.
  • a carrier is obtained that can deliver the substance and release the encapsulated substance in the early endosome.
  • it is stabilized in a pH region higher than pH 6.0 when it is used as a vesicle, and is designed to be destabilized at pH 6.0 for the first time to deliver encapsulated substances to late endosomes, and A carrier that can release the inclusion substance in the late endosome is obtained.
  • delivery of encapsulated material to the cytosol may cause cytotoxicity.
  • delivery of the antigen to the cytosol may inhibit cellular immunity induction.
  • the binding between the MHC class II molecule and the antigen leading to humoral immunity induction is considered to occur in the late endosome to lysosome. For this reason, when the carrier is decomposed in the lysosome and the antigen is released, it may not lead to effective antigen presentation.
  • Vesicles composed of the compounds of the present invention recognize the difference in acidity in the process from early endosome to late endosome to lysosome after being taken up by cells, and efficiently release inclusions at appropriate timing it can. Such a function can be useful for various applications. For example, it can help in developing vaccines that effectively induce humoral immunity. More details are as follows. In order to effectively induce humoral immunity, (a) antigen recognition in endosomes and lysosomes, (b) effective interaction of Toll-like receptors and antigens involved in immune activation, and (c ) It is considered necessary to induce high-efficiency binding of MHC molecules that perform antigen presentation and antigens.
  • the inclusion (antigen) is completely released at an early stage in the endosome, whereby the above-mentioned mutual The action can be efficiently caused and effective immunity induction can be performed.
  • a vesicle composed of a compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group containing a cyclohexyl group, a phenyl group, a pyridyl group or a naphthyl group exhibits responsiveness in a higher pH range. . For example, responsiveness at pH 6.0 or higher is shown. For this reason, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (A), a carrier capable of releasing inclusions in the initial endosome can be obtained.
  • vesicles composed of the compounds of the present invention are taken up by antigen-presenting cells (dendritic cells) in an extremely large amount, inclusions can be reliably released inside the endosome if they have the above characteristics. It is effective as a vaccine for inducing humoral immunity and as an immunosuppressant delivery system.
  • Vesicles composed of compounds of the present invention containing hydrocarbon groups show responsiveness in the lower pH range. For example, responsiveness at pH 5.0 or lower is shown. Therefore, by using a vesicle composed of the compound of the present invention containing a pH-responsive carboxyl group-containing hydrocarbon group (B), a carrier capable of releasing inclusions in lysosomes can be obtained.
  • a vesicle having pH responsiveness can be obtained.
  • the compound of the present invention can be obtained, for example, by adding R 3 and R 4 to polyamide dendron (DL) obtained as follows.
  • DL-G1 to DL-G4 indicate first to fourth generation polyamide dendrons, respectively.
  • DL-G0 is the 0th generation and does not have a dendron structure.
  • R 1 and R 2 are unsaturated hydrocarbon groups, they can be produced as follows. Oleylamine and oleyl chloride are reacted to synthesize oleyloleylamide. Next, dioleylamine is synthesized by hydride reduction, and a polyamidoamine dendron is synthesized by repeating a Michael addition reaction using methyl acrylate and an ester amide exchange reaction using ethylenediamine. This polyamidoamine dendron is represented as DL-G1-2C 18 -U2.
  • R ⁇ 1 > and R ⁇ 2 > is a saturated hydrocarbon group
  • Polyamideamine dendron is synthesized by repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine using dioctadecylamine as a starting material.
  • the polyamidoamine dendron denoted as DL-G1-2C 18.
  • R 3 and R 4 are performed as follows, for example. By reacting polyamidoamine dendron with 3-methylglutaric anhydride, a compound having —CO—CH 2 CH (CH 3 ) CH 2 —COOH as R 3 and R 4 is obtained.
  • composition of the present invention can suitably contain a phospholipid in addition to the compound of the present invention.
  • phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, plasmalogen, and phosphatidic acid.
  • phosphatidylethanolamine and phosphatidylcholine are preferably used alone or in combination.
  • the fatty acid residue of these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 18 carbon atoms.
  • lauroyl group myristoyl group , Palmitoyl group, stearoyl group, oleoyl group, linoleyl group and the like, and dioleoylphosphatidylethanolamine (DOPE) is particularly preferable.
  • DOPE dioleoylphosphatidylethanolamine
  • the blending amount of the phospholipid is not particularly limited, but when the total amount of the phospholipid and the compound of the present invention is 100 parts by weight, 30 to 90 parts by weight of the phospholipid, 70 to 10 parts by weight of the compound of the present invention, preferably phosphorous It is 50 to 80 parts by weight of lipid, 50 to 20 parts by weight of the compound of the present invention, more preferably 60 to 70 parts by weight of phospholipid, and 40 to 30 parts by weight of the compound of the present invention.
  • the compound of the present invention and the phospholipid may be present merely as a mixture, and the compound of the present invention and the phospholipid may be present. They may be combined to form a lipid membrane structure.
  • the existence form of the lipid membrane structure and the production method thereof are not particularly limited.
  • the existence form includes a dried lipid mixture form, a dispersed form in an aqueous solvent, and a dried form thereof. And a frozen form.
  • the dried lipid mixture can be produced, for example, by dissolving the lipid component to be used once in an organic solvent such as chloroform and then performing vacuum drying with an evaporator or spray drying with a spray dryer.
  • an organic solvent such as chloroform
  • Examples of the form in which the lipid membrane structure is dispersed in an aqueous solvent include monolayer liposomes, multilamellar liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, string micelles, and irregular layered structures And so on.
  • the size of the lipid membrane structure in a dispersed state is not particularly limited.
  • the particle diameter is 50 nm to several ⁇ m
  • the particle diameter Is from 5 nm to 50 nm.
  • the thickness per layer is 5 nm to 10 nm and these form a layer.
  • the composition of the aqueous solvent is not particularly limited, but in addition to water, sugar aqueous solutions such as glucose, lactose and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, and phosphate buffer And buffer solutions such as citrate buffer solution and phosphate buffered physiological saline solution, physiological saline, medium for cell culture, and the like.
  • sugar aqueous solutions such as glucose, lactose and sucrose
  • polyhydric alcohol aqueous solutions such as glycerin and propylene glycol
  • phosphate buffer And buffer solutions such as citrate buffer solution and phosphate buffered physiological saline solution, physiological saline, medium for cell culture, and the like.
  • the pH of the aqueous solvent From the viewpoint of chemical stability of the lipid, it is important to set the pH of the aqueous solvent from weakly acidic to neutral (pH 3.0 to 8.0) or to remove dissolved oxygen by nitrogen bubbling. . Further, when lyophilized storage or spray-dried storage is used, effective storage is possible by using a sugar aqueous solution, and when storing frozen, a sugar aqueous solution or a polyhydric alcohol aqueous solution is used.
  • the concentration of these aqueous solvent additives is not particularly limited.
  • an aqueous sugar solution 2 to 20% (W / V) is preferable, and 5 to 10% (W / V) is more preferable. preferable.
  • a polyhydric alcohol aqueous solution 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable.
  • the concentration of the buffer is preferably 5 to 50 mM, more preferably 10 to 20 mM.
  • the concentration of the lipid membrane structure in the aqueous solvent is not particularly limited.
  • the concentration of the total amount of phospholipid used as the lipid membrane structure is preferably 0.001 mM to 100 mM. More preferred is 01 mM to 20 mM.
  • the form in which the lipid membrane structure is dispersed in an aqueous solvent is produced by adding the dried lipid mixture to the aqueous solvent and further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. be able to. Moreover, it can also manufacture by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should not be specifically limited. When it is desired to control the size of the lipid membrane structure, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.
  • the compounds, compositions and kits of the present invention can be used for the uses described below.
  • the compounds, compositions and kits of the present invention are preferably used for introducing physiologically active substances into cells.
  • the cell may be a cell in the living body or a cell taken out of the living body. That is, the compound, composition and kit of the present invention are used for introducing a physiologically active substance into cells in vivo or in vitro.
  • the physiologically active substance is not particularly limited, and is, for example, a low molecular compound, a peptide or a gene.
  • a physiologically active substance encapsulated in a vesicle composed of the compound of the present invention can be contained in any of early endosomes, late endosomes or lysosomes. Can be released.
  • the peptide or gene is not particularly limited, but if it can be an antigen or can generate an antigen, it is encapsulated in a vesicle composed of the compound of the present invention and then introduced into an antigen-presenting cell. They can be released in early or late endosomes. This is preferable because it can be used for inducing antibody production by the humoral immunity response system through antigen presentation via MHC class II and thus treating the disease.
  • a gene capable of producing an antigen a gene used as a so-called DNA vaccine can be used as a so-called DNA vaccine can be used.
  • the DNA vaccine is a technique for inoculating DNA encoding antigen information as a vaccine.
  • a vector containing a DNA fragment encoding antigen information is usually used as a DNA vaccine.
  • vectors used as DNA vaccines contain unmethylated cytosine and guanine-rich sequence regions called unmethylated CpG motifs.
  • MAGE for the treatment of malignant melanoma
  • HER2 / neu for the treatment of breast cancer
  • CEA for the treatment of colorectal cancer
  • WT1 for the treatment of leukemia or various cancers. Etc. can be used.
  • Physiologically active substances that can act as immune inhibitors can be used. What can act as an immune inhibitor can be used regardless of whether the inhibitory action is selective or non-selective for a specific immune response.
  • the compound, composition and kit of the present invention of the present invention may be used for in vivo introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above diseases.
  • the compound or composition of the present invention may be administered to an individual together with a physiologically active substance.
  • the means for administration to an individual may be oral administration or parenteral administration, but parenteral administration is preferred.
  • the dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like. Examples of the dosage form for parenteral administration include injections, eye drops, ointments, suppositories and the like. Among these, an injection is preferable, and an administration method is preferably intravenous injection or local injection into a target cell or organ.
  • the compounds, compositions and kits of the present invention may be used for in vitro introduction into cells together with the physiologically active substance to be introduced for the purpose of treating the above-mentioned diseases.
  • a physiologically active substance is released in the endosome at an early stage after the vesicle is taken into the endosome, and effective immune induction is performed.
  • antibody production by the humoral immune system is induced in the cells through antigen presentation via MHC class II.
  • the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 20 parts by weight, preferably 3 to 15 parts by weight of the compound of the present invention with respect to 1 part by weight of the physiologically active substance. More preferably, 5 to 7 parts by weight are used.
  • the compounding ratio of the physiologically active substance and the compound of the present invention is 1 to 50 parts by weight, preferably 1 to 50 parts by weight, preferably 1 part by weight of the physiologically active substance. 5 to 30 parts by weight, more preferably 10 to 15 parts by weight is used.
  • any of oligonucleotide, DNA and RNA may be used.
  • a gene for introduction in vitro such as transformation
  • a gene which acts by expression in vivo for example, gene therapy
  • genes for use in breeding industrial animals such as laboratory genes and laboratory animals and livestock are preferred.
  • genes for gene therapy include genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes, and cytokines.
  • FBS Fetal Bovine Serum
  • DMEM Dulbecco's modified Eagle's medium
  • N N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical.
  • Pyranine was purchased from Tokyo Kasei Co., Ltd.
  • Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA).
  • Dioctadecylamine and calcein were purchased from Sigma.
  • Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
  • Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen.
  • DPX 2-mercaptoethanol
  • DPX Hoechst
  • Lysotracker Green DND-26 Lysotracker Red DND-99
  • Tf-Alexa555 Tf-Alexa555
  • As a dialysis membrane Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
  • DL-G1 and DL-G2 were synthesized using dioctadecylamine as a starting material by alternately performing a Michael addition reaction with methyl acrylate followed by an ester amide exchange reaction with ethylenediamine. This is the method reported by Tomalia et al. In the synthesis of dendrimers.
  • DL-G1-U2 reacts with oleylamine and oleyl chloride, and hydride reduction with lithium aluminum hydride (LiAlH 4 ), followed by Michael addition reaction with methyl acrylate and subsequent ester amide exchange reaction with ethylenediamine. To be synthesized.
  • the terminal amino groups of the synthesized DL-G1, DL-G2, and DL-G1-U2 were reacted with MDEG groups and EDEG groups activated with paranitrophenyl carbonate groups, and introduced at the ends to introduce MDEG-DL-G1, EDEG- DL-G1, MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
  • the peak near 0.88 ppm derived from the methyl group at the end of the octadecyl group or the methyl group at the end of the oleyl chain From the integration ratio of the peak near 0.96 ppm derived from, the integration ratio of the peak near 1.11 ppm derived from the terminal ethyl group of the introduced ethoxydiethylene glycol group, and the peak near 3.41 ppm, the ethoxydiethylene glycol group introduced into the dendrimer lipid Numbers were calculated. As a result, 2.0, 4.0, and 2.4 methoxydiethylene glycol groups were introduced at the ends of EDEG-DL-G1, EDEG-DL-G2, and EDEG-DL-G1-U2, respectively.
  • the first and second generation PAMAM dendron lipids having two alkyl chains and the first generation PAMAM dendron lipids having two oleyl chains were synthesized.
  • MDEG-DL-G1 and EDEG-DL-G1 which are various OEG group-binding dendron lipids in the first and second generation, by introducing various OEG groups with temperature responsiveness to their terminal amino groups MDEG-DL-G2, EDEG-DL-G2, MDEG-DL-G1-U2, and EDEG-DL-G1-U2 were synthesized.
  • dendron lipids are amphipathic molecules, it is thought that when they are dispersed in water, they form molecular aggregates, which causes temperature-responsive groups introduced into the hydrophilic region to accumulate on the aggregate surface. . And, it can be expected that the lipid molecule aggregate exhibits temperature responsiveness due to the interaction between the accumulated temperature responsive groups. Then, next, the temperature responsiveness of the lipid molecule assembly was examined using the synthesized temperature-responsive dendron lipid dispersion.
  • lipid dispersion The solvent was removed from each lipid chloroform solution using a rotary evaporator to form a lipid thin film.
  • a 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 2 mg / ml. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling.
  • the MDEG-DL-G2 Decrease in transmittance at around 68 ° C, EDEG-DL-G2 at around 32 ° C, MDEG-DL-G1-U2 at around 44 ° C, and EDEG-DL-G1-U2 at around 24 ° C. It was. These reductions in transmittance indicate that the molecular assembly has become unstable and aggregated.
  • the cloud point (Cloud Point)
  • the cloud points of -G1-U2 and EDEG-DL-G1-U2 were 34.2 ° C, 22.5 ° C, 67.6 ° C, 34.2 ° C, 45.4 ° C and 26.1 ° C, respectively. From the above, it was shown that temperature responsiveness can be imparted by integrating various OEG groups on the surface of the molecular assembly.
  • the cloud point temperature decreased as the end group became more hydrophobic, and the cloud point temperature increased as the generation number increased. This is considered to be because when the hydrophobicity is increased, aggregation due to hydrophobic interaction is likely to occur during dehydration due to temperature response. In addition, as the number of generations increases, the number of tertiary amines also increases and becomes more hydrophilic, so it is considered that the temperature response on the high temperature side was achieved. In addition, in the case of an alkyl chain and an oleyl chain, the cloud point of the molecular assembly of dendron lipids having an oleyl chain shifted to a higher temperature side. This is presumably because the lipid bilayer created by the molecular assembly is more fluid and has improved stability due to having a double bond.
  • FIG. 23 shows a part of the result at that time
  • FIG. 24 shows the result of plotting the cloudiness against pH.
  • the cloud point increased. This is because as the pH decreases, the degree of protonation of tertiary amino groups in polar groups increases and the hydrophilicity of the molecules increases, and electrostatic repulsion occurs between molecules and between molecules due to charge. Since the surface density of various OEG groups decreased, it was thought that cloud points were shown at higher temperatures. Further, when the pH became too high, the cloud point did not continue to decrease, but the cloud point was exhibited at a certain temperature. This is probably because the tertiary amino group in the polar group is no longer protonated.
  • the temperature response of the dendron lipid aggregate was evaluated by measuring the permeability of the lipid dispersion.
  • the transmittance decreased at a certain temperature or higher. This showed that the molecular assembly was aggregated.
  • the cloud point increased with a decrease in pH. This is considered to be because the protonation of the tertiary amine inside the dendron lipid was promoted and the molecular assembly became more hydrated.
  • Temperature control was performed using ETC-717. At this time, the temperature at which the transmittance dropped rapidly was taken as the cloud point. After the measurement, the pH of the solution was measured at room temperature, and the value was taken as the pH at the time of turbidity measurement.
  • FIG. 30 shows the result of plotting the cloud point against the ratio of introducing PEG lipid.
  • the cloud point increased. This is because the molecular assembly surface became more hydrophilic with the increase in PEG lipids, and the amount of PEG chains introduced on the surface increased, the surface density between EDEG groups decreased, and the interaction was reduced. This is considered to be due to the fact that the fluidity of the hydrophobic part of the molecular assembly was enhanced by the suppression and further the introduction of cholesterol.
  • FIG. 32 shows the results for EDEG-DL-G1 alone as a comparison.
  • the cloud point hardly changed. This is thought to be because the effect of increasing the hydrophilicity of the molecule due to the protonation of the tertiary amino group in the polar group accompanying the decrease in pH was slight compared to the hydration of the surface by the PEG chain. . It was also found that at any pH, the PEG lipid introduced showed a cloud point on the higher temperature side.
  • FIG. 34 shows the results of morphological observation of lipid molecular aggregates (pH 7.4). At 10 ° C. below the cloud point, a spherical molecular assembly having a particle size of about 150 to 200 nm was observed (FIG. 34 (A)). This aggregate is considered to have a vesicle structure because of its particle size.
  • the temperature responsiveness evaluation of the EDEG-DL-G1 / PEG-Chol lipid molecular assembly was performed by measuring the permeability of the lipid dispersion.
  • the transmittance decreased rapidly above a certain temperature. This showed that the molecular assembly was aggregated. That is, it was found that the introduction of PEG lipid did not impair the temperature response function.
  • the introduction rate of PEG lipid was increased, the cloud point of the molecular assembly increased. This is probably because the introduction of PEG-Chol hydrated the surface of the molecular assembly and required more energy to dehydrate and transfer.
  • the particle size was about 200 nm, and when the temperature was above the cloud point, the particle size increased. This is because when the temperature is below the cloud point, the molecular aggregate is hydrated and stable while maintaining a certain particle size, and when the cloud point is exceeded, the hydrophilicity / hydrophobicity balance becomes unstable due to dehydration due to temperature response. This is probably because the hydrophobic interaction worked to form aggregates.
  • FBS Fetal Bovine Serum
  • Dulbecco's modified Eagle's medium (DMEM) was purchased from Nissui Pharmaceutical.
  • Disodium hydrogen phosphate, potassium dihydrogen phosphate, benzylpenicillin potassium, streptomycin sulfate, acetyl CoA, ampicillin sodium, chloramphenicol, ethidium bromide were purchased from Wako Pure Chemical.
  • Calcium chloride, magnesium chloride hexahydrate, potassium chloride, trishydroxymitylaminomethane (Tris), and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Cell lysing agent Luc-PGC-50 was purchased from Toyo Ink.
  • Sodium chloride was purchased from Nacalai Tesque. Trypsin was purchased from DIFCO LABORATORIES (USA).
  • Rhodamine-PE (0.6 mol%) was added to a chloroform solution in which EDEG-DL-G1 and PEG lipids were mixed at a ratio of 95/5, and the solvent was removed using a rotary evaporator. Was removed to form a lipid film.
  • a 10 mM phosphate buffer solution was added thereto to adjust the pH to 3.0 and a concentration of 1.0 mM. This was irradiated with ultrasonic waves at 45 ° C. for 10 minutes using a bath-type ultrasonic irradiation apparatus, and then allowed to stand at 45 ° C. for 20 minutes. Then, it was left still for 60 minutes or more under ice cooling. Finally, the pH was adjusted to 7.4 to prepare a dispersion.
  • the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
  • the EDEG-DL-G1 / PEG-Chol lipid molecular assembly into which the constructed PEG lipid was introduced was labeled with a fluorescent label, and the delivery function of the vesicle in the cell was evaluated.
  • the vesicle surface is hydrophilic before showing a temperature response, so it only weakly adsorbs to the cell surface, but when the cloud point is exceeded, the vesicle dehydrates due to the temperature response and changes its shape. Therefore, it is thought that it was taken in in the cell. At this time, the vesicle changes its shape, interacts strongly with the cell membrane, is efficiently taken up into the cell by endocytosis, or moves through the cell membrane directly by hydrophobic interaction. It is thought that it was taken in.
  • Dendron lipid / PEG-Chol aggregate dispersions (10 M phosphoric acid, 140 M NaCl, pH3.0) obtained by dispersing mixed thin films of MDEG-G1 and PEG-Chol (PEG molecular weight 1000) in various ratios
  • the temperature responsiveness was evaluated by measuring the temperature dependence of the transmittance in addition to a buffer of 10 mM mM phosphoric acid, 140 mM mM NaCl, and pH 7.4. Temperature increase rate 4 °C / min.
  • the cloud point of MDEG-G1 / PEG-Chol aggregate increased with increasing PEG-Chol content. It was found that the response temperature (cloud point) of the aggregate can be adjusted by adjusting the PEG-Chol content. It was also found that a dendron lipid aggregate having a cloud point near 40 ° C. can be obtained by containing 2% of PEG-Chol.
  • HeLa cells derived from human cervical cancer
  • HeLa cells were seeded in a 12-well dish at 100,000 / well and cultured for 24 hours. Wash twice with PBS (+) and once with PBS (-), add 500 ⁇ L of DMEM (without serum), and add 500 ⁇ L of dendron lipid / PEG-Chol aggregate dispersion to a lipid concentration of 0.5 mM.
  • the cells were incubated at 37 ° C., 42 ° C. and 44 ° C. for 15 minutes and 30 minutes in a CO 2 incubator. Thereafter, the cells were washed twice with PBS (+) and once with PBS (-), and the cells were detached with trypsin.
  • the MDEG-DL-G1-2C 18 / PEG-Chol (98/2) aggregate did not change much even when the temperature and contact time were changed. This is because the dendron lipid MDEG-DL-G1-2C 18 terminal group has a lower hydrophobicity than EDEG-DL-G1-2C 18 , and so the interaction with the cell works very strongly even when morphological changes occur. It is thought that there was not.
  • temperature-responsive groups are given temperature responsiveness by introducing oligoethylene glycol chains with excellent biocompatibility as temperature-responsive groups into polar groups of dendron lipids and accumulating them on the surface of the molecular assemblies. It was confirmed that it was possible. Also, the cloud point temperature was different depending on the generation and end group. This is considered due to the balance between hydrophobicity and hydrophilicity in the molecular structure. Therefore, it is considered that it is possible to construct a molecular assembly that responds at a desired temperature by designing the balance between the hydrophobic part and the hydrophilic part in the molecular structure.
  • the temperature-responsive dendron lipid aggregate into which polyethylene glycol lipid is introduced changes its shape dramatically above the cloud point near body temperature, and has both high stability and biocompatibility due to the effect of polyethylene glycol lipid.
  • a vesicle was constructed. This PEG-Chol complex type dendron lipid vesicle can control the delivery function into cells by temperature.
  • Second invention Preparation of compounds of the invention wherein R 1 and R 2 are unsaturated hydrocarbon groups
  • FBS Fetal Bovine Serum
  • DMEM Dulbecco's modified Eagle's medium
  • N N-dimethylformamide, tetrahydrofuran, petroleum ether, sodium cyanide, lithium aluminum hydride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium benzylpenicillin, and streptomycin sulfate were purchased from Wako Pure Chemical.
  • Pyranine was purchased from Tokyo Kasei Co., Ltd.
  • Chloroform, ethyl acetate, methanol, n-hexane, sodium sulfate, calcium chloride, magnesium chloride hexahydrate, potassium chloride, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Kishida Chemical.
  • Triethylamine, methyl acrylate, ethylenediamine, diethyl ether, sodium chloride, RPMI-1640 liquid medium, and MEM non-essential amino acid solution were purchased from Nacalai Tesque. Trypsin was purchased from DIFCOLABORATORIES (USA).
  • Dioctadecylamine and calcein were purchased from Sigma.
  • Oleylamine, oleoyl chloride, cyclohexanedicarboxylic anhydride and 3-methylglutaric anhydride were purchased from ALDRICH.
  • Dichloromethane was purchased from Sigma-ALDRICH®. 2-mercaptoethanol, DPX, Hoechst, Lysotracker Green DND-26, Lysotracker Red DND-99, and Tf-Alexa555 were purchased from Invitrogen.
  • DPX 2-mercaptoethanol
  • DPX Hoechst
  • Lysotracker Green DND-26 Lysotracker Red DND-99
  • Tf-Alexa555 Tf-Alexa555
  • As a dialysis membrane Spectra / Por 6 (fractional molecular weight 2000, FE-0526-33) was purchased from Spectrum Laboratories Inc. Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography.
  • DL-G1 by synthesizing oleyl oleoylamide with oleylamine and oleoyl chloride, then synthesizing dioleylamine by hydride reduction, repeating Michael addition reaction using methyl acrylate and ester amide exchange reaction using ethylenediamine -2C 18 -U2 was synthesized. This is the method reported by Tomalia et al. In the synthesis of dendrimers. Further, CHexDL-U2 and MGluDL-U2 were synthesized by reacting the synthesized DL-G1-2C 18 -U2 with 3-Methylglutaric Anhydride or Cyclohexanedicarboxylic Anhydride.
  • MGluAn 3-Methylglutaric Anhydride
  • DMF 3 mL
  • 430 mg (0.909 mmol) of DL-G1-2C 18 -U2 was dissolved in 4 mL of DMF and mixed, 0.370 mL of triethylamine (TEA) was added, and the mixture was stirred at 50 ° C. for 7 days under a nitrogen atmosphere.
  • TEA triethylamine
  • a dendron lipid solution in chloroform was spread on it and left to stand for 20 minutes to blow off the chloroform.
  • LB membrane measurement device In ⁇ -A mode, the surface pressure of the lipid monolayer was measured with a pressure gauge when the molecular surface area of the lipid surface on the solvent surface was reduced by laterally compressing the solvent surface with a barrier. .
  • the solvent on the trough was collected, the trough and the barrier were washed with ethanol, and the solvent was developed.
  • CHex-DL-G1-2C 18 -U2 liposome (hereinafter referred to as UCG1) is prepared by mixing a predetermined amount of EYPC (10 mg / ml) in chloroform and a predetermined amount of CHex-DL-G1-2C 18 -U2 (3.65 mg / ml). ml) of chloroform solution was mixed and the solvent was removed by a rotary evaporator to form a thin film, followed by vacuum drying for 4 hours to completely remove the solvent.
  • EYPC liposomes a predetermined amount of EYPC in chloroform was added, the solvent was removed with a rotary evaporator to form a thin film, and the solvent was completely removed by vacuum drying for 4 hours.
  • the particle size and ⁇ potential of liposomes were determined by a dynamic light scattering method.
  • the liposome solution was added to PBS (-) or 0.1 mM phosphate buffer (final volume 2.5 ml) adjusted to each pH so that the lipid concentration in the cell was 0.1 mM.
  • the mixture was allowed to stand for 15 minutes, and the particle size and ⁇ potential were measured.
  • the measurement was performed at 25 ° C., and was measured using ELS-8000F manufactured by Otsuka Electronics Co., Ltd.
  • Liposomes are dispersed in PBS (-) pH 7.4 so that the lipid concentration is 0.5 mM, and the pH is continuously changed at 37 ° C.
  • the transmittance of light at 500 nm was measured using a V-560 type ultraviolet / visible spectrophotometer manufactured by JASCO Corporation.
  • mice-derived cell culture strain DC2.4 cells Mouse-derived cell culture strain DC2.4 cells Is RPMI-1640 medium containing 10% FBS, 0.1 mg / ml benzylpenicillin potassium, 0.1 mg / ml streptomycin sulfate, 2 mM L-glutamine, 0.1 mM MEM non essential amino acid solution and 0.55 mM 2-mercaptoethanol.
  • the cells were cultured in a CO 2 incubator (MCO-96) manufactured by Sanyo Electric Co. at a CO 2 concentration of 5% at 37 ° C.
  • the cells After washing 3 times with HBSS, the cells are detached using 300 ⁇ l of trypsin aqueous solution (trypsin (DIFCO) 250 mg, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml) per well, and a flow cytometer It collected in the tube for use.
  • trypsin (DIFCO) 250 mg
  • EDTA disodium ethylenediaminetetraacetate
  • PBS 100 ml a flow cytometer
  • HeLa cells were seeded at 1 ⁇ 10 5 per well of a 12-well dish and cultured at 37 ° C for 48 hours in 0.5 ml of 10% FBS-containing DMEM medium. . Then, after washing twice with PBS containing 0.36 mM CaCl 2 and 0.42 mM MgCl 2 (PBS (+)), 0.5 ml of DMEM medium containing 10% FBS was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM, and incubated at 37 ° C. for 4 hours. Then, wash twice with PBS (+) and once with PBS (-) (PBS without Ca2 + and Mg2 +).
  • trypsin aqueous solution 250 mg trypsin (DIFCO) per well, disodium ethylenediaminetetraacetate (EDTA) 1 mg, PBS 100 ml
  • EDTA disodium ethylenediaminetetraacetate
  • Hela cells are seeded on a Matsunami glass bottom dish at 2 ⁇ 10 5 cells per plate, in 10 ml FBS-containing DMEM medium at 37 ° C. Incubated overnight. Thereafter, after washing twice with PBS (+) and once with PBS ( ⁇ ), 1.0 ml of 10% FBS-containing DMEM medium was added. The liposome solution was added to each well so that the lipid concentration was 0.5 mM (total volume: 2 mL), and incubated at 37 ° C. for 4 hours or 24 hours. Then, after washing twice with PBS (+) and once with PBS ( ⁇ ), OPTI-MEM was added, and intracellular dynamics were observed with a confocal laser microscope (LSM5 EXCITER (ZEISS)).
  • LSM5 EXCITER confocal laser microscope
  • Lysotracker staining of late endosomes / lysosomes Take 1 ⁇ L of Lysotracker (Invitrogen, Lysotracker Green DND-26, Lysotracker RedDND-99, DMSO solution: 1 mM) and RPMI 1640 or DMEM without FBS A staining solution was prepared by adding 99 ⁇ L of medium. Liposomes were taken up by the method described above, and then 1985 ⁇ L of RPMI 1640 or DMEM medium not containing FBS and 15 ⁇ L of staining solution were added and incubated for 5 minutes. Thereafter, after washing three times, PBS was added again, and intracellular dynamics were observed with a confocal laser microscope (LSM 5EXCITER (ZEISS)).
  • LSM 5EXCITER confocal laser microscope
  • a desired responsiveness can be obtained by constructing a vesicle by appropriately combining two or more compounds of the present invention having different pH responsiveness. It was verified as follows whether or not vesicles having the same could be obtained.
  • pH -responsive dendron lipids could be synthesized by the appearance of peaks derived from the introduced terminal groups.
  • MGluDL-U2 the peak derived from the methyl group of 3-methylglutaric acid (6H) near 1.03 ppm appeared, and in CHexDL-U2 and CHexDL-S, the peak derived from cyclohexanedicarboxylic acid (16H) at 1.2-2.0 ppm It was confirmed that synthesis was possible by the appearance of.
  • mass spectrometry was performed in the ESI-negative mode, it was confirmed that the synthesis was surely performed because it showed a value equivalent to the theoretical value.
  • FIG. 58 shows a plot of the surface area for each pH when the surface pressure is 25 mN / m.
  • CHexDL-U2 tended to have a larger area at the same surface pressure than MGluDL-U2. This is because CHexDL-U2 has a larger excluded volume of terminal groups. However, there is no clear difference between these two types. Since it is considered that there is no clear difference in G1 because only two terminal groups can be introduced, it is considered that this difference in terminal groups will occur by increasing the number of generations.
  • the area of the saturated type was larger at the same surface pressure (Fig. 59). Further, when compared at the same surface pressure, the surface area was larger in the case of acidity than neutrality. As shown in FIG. 60, this is thought to be due to the fact that the pH group decreases and the carboxy group is protonated to be distributed to the gas-liquid interface. Furthermore, when the internal tertiary amine is protonated, intermolecular electrostatic repulsion is also generated, and the surface area is thought to have increased when compared at the same surface pressure.
  • SCG1 liposomes The effect of pH on the pyranine release of UCG1 liposomes and saturated CHexDL liposomes (hereinafter SCG1 liposomes) was examined.
  • the prepared UMG1, UCG1, and SCG1 liposomes had the following lipid composition ratios.
  • release does not occur over time in a high pH environment, whereas inclusions are released from the liposomes over time in a low pH environment.
  • the inclusion release under neutrality did not occur so much, but the inclusion release occurred in any liposome under weak acidity.
  • the maximum release rate from liposomes was about 30% for UMG1, 90% or more for UCG1, and about 60% for SCG1.
  • the maximum value of the release rate of inclusions differs between MGlu -introduced and CHex -introduced, which is thought to be due to the difference in the hydrophobicity of the end groups. That is, it is considered that the CHex ⁇ ⁇ group having a higher degree of hydrophobicity interacts more strongly with the membrane, whereas the MGlu ⁇ group having a lower degree of hydrophobicity has a weak interaction with the membrane, so that the release rate remains low.
  • the reason why the release rate of inclusions is different between the saturated type and the unsaturated type even when the same CHex is introduced is that the saturated dendron has an alkyl chain (octadecyl chain) which is a hydrophobic site as an unsaturated type. It has high crystallinity compared to the oleyl chain, and it is thought that in the saturated type, dendron lipids are separated and easily assembled in the lipid membrane. It is thought that the lipid membrane became partially crystal-like, and the liposome membrane became hard and leakage of inclusions did not occur easily.
  • alkyl chain octadecyl chain
  • the release rate was maximized when 25% of the dendron lipid was modified on the liposome, and no further improvement in the release rate was observed even when the amount of the dendron lipid charged was increased.
  • the effect of the dendron lipid end group on the liposome lipid membrane is considered to be sufficiently achieved by 25% modification.
  • the amount of dendron lipid charged was 50%, UMG1 dissolved, aggregates precipitated in UCG1 and SCG1 liposomes, and liposomes were not formed. At this time, it is considered that MGluDL-U2 has reached micelle formation and dissolved, and CHexDL-U2 and CHexDL-S have self-aggregated dendron lipids.
  • the particle size was about 100 nm under neutral and weak acidity, but the particle size increased around pH5.0 and became maximum at pH4.5, and when pH was further lowered, it was again 100 nm. The particles became about the size. Also in turbidity measurement, the transmittance starts to decrease at around pH 5.0 mm, the transmittance becomes the lowest at pH 4.4 to pH 4.5 mm, and the permeability is again about the same as under neutral by lowering the pH value. Value. From this, in UMG1UM liposomes, when pH is lowered, the interaction between the hydrophobic part of its own liposome membrane and the hydrophobic part of the end group does not occur so much, and it is thought that aggregation interacting with other liposomes occurs .
  • EYPC liposome ⁇ potential did not change greatly depending on pH, and was almost neutral.
  • the effect of pH on the zeta potential of UMG1 ⁇ liposome, UCG1 ⁇ liposome and SCG1 ⁇ liposome was investigated. All liposomes had negatively similar values when weakly basic, the ⁇ potential increased with decreasing pH, and were positively similar values under acidic conditions.
  • the ⁇ potential was positive at pH 5-6, and in the UCG1 and SCG1 liposomes, it was already positive near neutrality.
  • the ⁇ potential of EYPC liposomes does not change even when the pH is changed, it is considered that the change in the ⁇ potential of the liposome occurred because the charge state of the pH responsive dendron lipid was changed. Therefore, the difference in the ⁇ potential depending on the pH of the liposome can be explained as follows from the difference in the charged state of the dendron lipid. It is considered that the charge state of the dendron lipid at each pH of the UMG1 liposome is as shown in FIG.
  • the terminal carboxy group Under neutral and weak basicity, the terminal carboxy group has a negative charge, so the ⁇ potential is considered to be negative.
  • the ⁇ potential since protonation of tertiary amines inside dendron occurs at pH 6.0 to pH 7.0%, protonation of tertiary amino groups inside dendron lipids occurs at pH 5.5 to pH 7.0%. It is considered that the ⁇ potential became neutral at around pH 5.5.
  • the terminal carboxy group is protonated, and the ⁇ potential is considered to be a positive value.
  • the charge state of the pH-responsive dendron lipid at each pH of the UCG1 and SCG1 liposomes is considered as shown in FIG.
  • the zeta potential is already positive. Since the protonation of the tertiary amino group in the dendron occurs at pH 6.0 to pH 7.0, the increase in ⁇ potential up to pH 7.0 is thought to be due to the protonation of the terminal carboxy group. . Further, it is considered that the ⁇ potential became positive due to the protonation of the tertiary amino group in the dendron due to a further decrease in pH.
  • FIG. 31 shows the results for MGluDL-U2
  • FIG. 32 shows the results for CHexDL-U2
  • FIG. 33 shows the results for CHexDL-S.
  • the ⁇ potential is negative and maximum, and the area is large.
  • the ⁇ potential increased, and the area area was minimized in the unsaturated type, and the area area was also decreased in the saturated type. This is considered to be due to the fact that the repulsion of the dendron portion of the dendron lipid became neutral due to the decrease in the ⁇ potential, so that charge repulsion disappeared and the dendrons packed more closely.
  • the pH value decreased further, the area of the area increased as the ⁇ potential increased. This is because the dendron site has been charged again. As described above, it was found that the area of the area also changed in synchronization with the change in the ⁇ potential.
  • FIGS. 73-75 show the results for UMG1.25, UCG1.25 and SCG1.25, respectively.
  • the ⁇ potential is negative and no release occurs.
  • release occurs when the ⁇ potential rises (the charge state of the dendron lipid changes), and that the inclusion release is also maximized in the pH range where the ⁇ potential is positive and maximum.
  • UMG1 has a low release rate, making it difficult to confirm the correlation, but this is true for all liposomes.
  • the carboxy group at the end of the dendron lipid is negatively charged under weak basicity, and since it does not interact with the liposome membrane, the liposome membrane is considered to be stably held.
  • the dendron under neutral conditions, from the results of monomolecular film measurement, the dendron can be packed more densely, so the inclusion is considered to be stably held.
  • the pH decreases, the hydrophobic interaction of the terminal membrane derived from 3-methylglutaric acid or 1.2-cyclohexanedicarboxylic acid increases due to the protonation of the carboxy group at the end of the dendron lipid, increasing the hydrophobic interaction with the liposome membrane.
  • the liposome membrane is destabilized by the formation of a hydrophilic site in the liposome membrane, which was hydrophobic in basic and neutralization, due to the protonation of the tertiary tertiary amino group inside the dendron lipid. It is thought that the membrane was destabilized due to the packing of lipid molecules (egg yolk phosphatidylcholine) entering the pH -responsive dendron lipid end group, resulting in destabilization of the membrane and release.
  • lipid molecules egg yolk phosphatidylcholine
  • UMG1.25 liposomes Compared to the amount of EYPC® liposomes taken into cells, the amount of UMG1.25 liposomes was slightly less, and the amount of UCG1.25 and SCG1.25 liposomes was 3-5 times higher.
  • uptake after adsorption due to hydrophobic interaction with the cell membrane may be considered. From the comparison of ⁇ potential under physiological conditions of these liposomes, this difference is considered as follows. Since the surface of the cell membrane is negatively charged, UMG1.25, which had a negative ⁇ potential, has little uptake due to electrostatic repulsion, and the hydrophobicity of the dendron end group is not so high. Uptake by hydrophobic interaction with can not be expected.
  • UMG1.25 liposomes with negative ⁇ potential had the least uptake.
  • the terminal group (MGlu group) of MGluDL-U2 incorporated in the liposome is not so large in excluded volume (steric hindrance), and the hydrophobicity is not so high. From this, it is thought that the interaction (electrostatic interaction or hydrogen bond) between the terminal carboxy group and the hydrophilic site on the outside of the liposome occurred, and it seems that it was in a state of being bound to the choline component in the liposome. It is done.
  • FIGS. Show. 77 to 80 show the results for DC 2.4 cells
  • FIGS. 81 to 85 show the results for Hella cells. 38 and 42 are 4 hours after the addition of SCG1.25, FIGS. 39 and 43 are 4 hours after the addition of UCG1.25, and FIGS. 40 and 83 are 4 after the addition of UMG1.25.
  • FIGS. 80 and 84 are the results 4 hours after the addition for EYPC
  • FIG. 85 are the results 24 hours after the addition for UMG1.25.
  • Rhodoamine- labeled liposomes and late endosome / lysosomal calcein-encapsulated UCG1.25 liposomes were incorporated, and late endosome / lysosomes were stained.
  • the results are shown in FIG.
  • the lyso tracker was red. This figure shows that the positions of late endosomes / lysosomes (intracellular acidic vesicles) and released calcein coincide.
  • CHexDL-G2-2C 18 hereinafter sometimes CHexDL-G1-2C 18 and CHexDL-G2-2C 18, respectively referred to as CHexDL-G1-S and CHexDL-G2-S.
  • CHexDL-G2-S Various evaluations were performed on CHex-DL-G2-S as follows. In the following, unless otherwise described, the same operation as described for CHex-DL-G1-S was performed.

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Abstract

La présente invention s'attaque au problème consistant à améliorer l'équilibre entre (1) la réactivité à la température, (2) la stabilité in vivo et (3) la biocompatibilité d'un support sensible à la température qui est utilisé dans le but d'améliorer la directivité vis-à-vis d'une cible d'un médicament. La présente invention propose, comme solution pour le problème, un composé qui est représenté par l'une des formules DL-G1 à DL-G4 suivantes. DL-G1:R1R2NX(XHR3)XHR4 DL-G2:R1R2NX(X(XHR3)XHR4)2 DL-G3:R1R2NX(X(X(XHR3)XHR4)2)2 DL-G4:R1R2NX(X(X(X(XHR3)XHR4)2)2)2
PCT/JP2013/063459 2012-05-14 2013-05-14 Composé fonctionnel, assemblage moléculaire contenant un composé fonctionnel, composition contenant un assemblage moléculaire, trousse et utilisation de l'assemblage moléculaire, de la composition ou du trousse WO2013172358A1 (fr)

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JP2012110911 2012-05-14

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WO2018164116A1 (fr) * 2017-03-06 2018-09-13 国立大学法人筑波大学 Liposome, agent anticancéreux et kit de thérapie anticancéreuse

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JP2007137805A (ja) * 2005-11-16 2007-06-07 Osaka Prefecture Univ ポリアミドアミンデンドロン脂質を含む遺伝子等運搬媒体組成物
WO2008139855A1 (fr) * 2007-05-08 2008-11-20 Osaka Prefecture University Public Corporation Lipide portant des dendrimères polyamidoamine contenant un groupe acyle de faible poids moléculaire
JP2010505873A (ja) * 2006-10-03 2010-02-25 アルナイラム ファーマシューティカルズ インコーポレイテッド 脂質含有製剤
JP2011502134A (ja) * 2007-11-05 2011-01-20 セルシオン コーポレイション 治療薬を含有する新規な温度感受性リポソーム

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JP2007137805A (ja) * 2005-11-16 2007-06-07 Osaka Prefecture Univ ポリアミドアミンデンドロン脂質を含む遺伝子等運搬媒体組成物
JP2010505873A (ja) * 2006-10-03 2010-02-25 アルナイラム ファーマシューティカルズ インコーポレイテッド 脂質含有製剤
WO2008139855A1 (fr) * 2007-05-08 2008-11-20 Osaka Prefecture University Public Corporation Lipide portant des dendrimères polyamidoamine contenant un groupe acyle de faible poids moléculaire
JP2011502134A (ja) * 2007-11-05 2011-01-20 セルシオン コーポレイション 治療薬を含有する新規な温度感受性リポソーム

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TOSHINARI TAKAHASHI ET AL.: "Synthesis of Poly(amidoamine) Dendron-Bearing Lipids with Poly(ethylene glycol) Grafts and Their Use for Stabilization of Nonviral Gene Vectors", BIOCONJUGATE CHEMISTRY, vol. 18, no. 4, 2007, pages 1163 - 1169 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018164116A1 (fr) * 2017-03-06 2018-09-13 国立大学法人筑波大学 Liposome, agent anticancéreux et kit de thérapie anticancéreuse

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